Nomadic enhancers: tissue-specific cis-regulatory elements of yellow have divergent genomic positions among Drosophila species

cis-regulatory DNA sequences known as enhancers control gene expression in space and time. They are central to metazoan development and are often responsible for changes in gene regulation that contribute to phenotypic evolution. Here, we examine the sequence, function, and genomic location of enhancers controlling tissue- and cell-type specific expression of the yellow gene in six Drosophila species. yellow is required for the production of dark pigment, and its expression has evolved largely in concert with divergent pigment patterns. Using Drosophila melanogaster as a transgenic host, we examined the expression of reporter genes in which either 5' intergenic or intronic sequences of yellow from each species controlled the expression of Green Fluorescent Protein. Surprisingly, we found that sequences controlling expression in the wing veins, as well as sequences controlling expression in epidermal cells of the abdomen, thorax, and wing, were located in different genomic regions in different species. By contrast, sequences controlling expression in bristle-associated cells were located in the intron of all species. Differences in the precise pattern of spatial expression within the developing epidermis of D. melanogaster transformants usually correlated with adult pigmentation in the species from which the cis-regulatory sequences were derived, which is consistent with cis-regulatory evolution affecting yellow expression playing a central role in Drosophila pigmentation divergence. Sequence comparisons among species favored a model in which sequential nucleotide substitutions were responsible for the observed changes in cis-regulatory architecture. Taken together, these data demonstrate frequent changes in yellow cis-regulatory architecture among Drosophila species. Similar analyses of other genes, combining in vivo functional tests of enhancer activity with in silico comparative genomics, are needed to determine whether the pattern of regulatory evolution we observed for yellow is characteristic of genes with rapidly evolving expression patterns.     

Comparison of the gap segmentation gene hunchback between Drosophila melanogaster and Drosophila virilis reveals novel modes of evolutionary change.

We have cloned and sequenced a large portion of the hunchback (hb) locus from Drosophila virilis. Comparison with the Drosophila melanogaster hb sequence shows multiple strong homologies in the upstream and downstream regions of the gene, including most of the known functional parts. The coding sequence is highly conserved within the presumptive DNA-binding finger regions, but more diverged outside of them. The regions of high divergence are correlated with regions which are rich in short direct repeats (regions of high 'cryptic simplicity'), suggesting a significant influence of slippage-like mechanisms in the evolutionary divergence of the two genes. Staining of early D.virilis embryos with an hb antibody reveals conserved and divergent features of the spatial expression pattern at blastoderm stage. It appears that the basic expression pattern, which serves as the gap gene function of hb, is conserved, while certain secondary expression patterns, which have separate functions for the segmentation process, are partly diverged. Thus, both slippage driven mutations in the coding region, which are likely to occur at higher rates than point mutations and the evolutionary divergence of secondary expression patterns may contribute to the evolution of regulatory genes.     

Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns

Body coloration affects how animals interact with the environment. In insects, the rapid evolution of black and brown melanin patterns suggests that these are adaptive traits. The developmental and molecular mechanisms that generate these pigment patterns are largely unknown. We demonstrate that the regulation and function of the yellow and ebony genes in Drosophila melanogaster play crucial roles in this process. The Yellow protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. Conversely, Ebony is required to suppress some melanin formation, and is expressed in cells that will produce both melanized and non-melanized cuticle. Ectopic expression of Ebony inhibits melanin formation, but increasing Yellow expression can overcome this effect. In addition, ectopic expression of Yellow is sufficient to induce melanin formation, but only in the absence of Ebony. These results suggest that the patterns and levels of Yellow and Ebony expression together determine the pattern and intensity of melanization. Based on their functions in Drosophila melanogaster, we propose that changes in the expression of Yellow and/or Ebony may have evolved with melanin patterns. Consistent with our hypothesis, we find that Yellow and Ebony are expressed in complementary spatial patterns that correlate with the formation of an evolutionary novel, male-specific pigment pattern in Drosophila biarmipes wings. These findings provide a developmental and genetic framework for understanding the evolution of melanin patterns.     

Evolution of the Autosomal Chorion Locus in Drosophila. I. General Organization of the Locus and Sequence Comparisons of Genes S15 and S19 in Evolutionarily Distant Species

We have isolated clones corresponding to the autosomal chorion locus of Drosophila melanogaster, from two distantly (D. virilis and D. grimshawi) and one closely (D. subobscura) related species. In all the species the locus is unique within the genome and encompasses the same four chorion genes and an adjacent nonchorion gene, in the same order. In all species the locus specifically amplifies in the ovary, as in D. melanogaster. We present the nucleotide sequences of DNA segments that total 8.3 kb in length and include gene s15-1 from D. subobscura, D. virilis, and D. grimshawi as well as gene s19-1 from D. subobscura and D. grimshawi. They show clearly nonuniform rates of divergence, both within and outside the limits of the genes. Highlighted by a background of extensive sequence divergence elsewhere in the extragenic region, highly conserved elements are observed in the 5' flanking DNA and might represent regulatory elements.     

Evolution of cis-regulatory sequence and function in Diptera

Cis-regulatory sequences direct patterns of gene expression essential for development and physiology. Evolutionary changes in these sequences contribute to phenotypic divergence. Despite their importance, cis-regulatory regions remain one of the most enigmatic features of the genome. Patterns of sequence evolution can be used to identify cis-regulatory elements, but the power of this approach depends upon the relationship between sequence and function. Comparative studies of gene regulation among Diptera reveal that divergent sequences can underlie conserved expression, and that expression differences can evolve despite largely similar sequences. This complex structure-function relationship is the primary impediment for computational identification and interpretation of cis-regulatory sequences. Biochemical characterization and in vivo assays of cis-regulatory sequences on a genomic-scale will relieve this barrier.     

Evolutionary conservation of regulatory strategies for the sex determination factor transformer-2.

Sex determination in Drosophila melanogaster is regulated by a cascade of splicing factors which direct the sex-specific expression of gene products needed for male and female differentiation. The splicing factor TRA-2 affects sex-specific splicing of multiple pre-mRNAs involved in sexual differentiation. The tra-2 gene itself expresses a complex set of mRNAs generated through alternative processing that collectively encode three distinct protein isoforms. The expression of these isoforms differs in the soma and germ line. In the male germ line the ratio of two isoforms present is governed by autoregulation of splicing. However, the functional significance of multiple TRA-2 isoforms has remained uncertain. Here we have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative mRNAs produced in D. virilis testes suggests that germ line-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster. The similarity in structure and function of the tra-2 genes in these divergent Drosophila species supports the idea that sexual differentiation in D. melanogaster and D. virilis is accomplished under the control of similar regulatory pathways.     

Divergence of the Yellow Gene between Drosophila Melanogaster and D. Subobscura: Recombination Rate, Codon Bias and Synonymous Substitutions

The yellow (y) gene maps near the telomere of the X chromosome in Drosophila melanogaster but not in D. subobscura. Thus the strong reduction in the recombination rate associated with telomeric regions is not expected in D. subobscura. To study the divergence of a gene whose recombination rate differs between two species, the y gene of D. subobscura was sequenced. Sequence comparison between D. melanogaster and D. subobscura revealed several elements conserved in noncoding regions that may correspond to putative cis-acting regulatory sequences. Divergence in the y gene coding region between D. subobscura and D. melanogaster was compared with that found in other genes sequenced in both species. Both, yellow and scute exhibit an unusually high number of synonymous substitutions per site (p(s)). Also for these genes, the extent of codon bias differs between both species, being much higher in D. subobscura than in D. melanogaster. This pattern of divergence is consistent with the hitchhiking and background selection models that predict an increase in the fixation rate of slightly deleterious mutations and a decrease in the rate of fixation of slightly advantageous mutations in regions with low recombination rates such as in the y-sc gene region of D. melanogaster.     

Changes in the Pattern of Twisted Gastrulation Gene Expression Among Drosophila Species

A long-standing hypothesis posits that morphological changes may be more likely to result from changes in regulation of gene expression than from changes in the protein coding sequences of genes. We have compared the expression pattern of the twisted gastrulation (tsg) gene among five Drosophila species: D. melanogaster, D. simulans, D. subobscura, D. mojavensis, and D. virilis. The tsg gene encodes a secreted protein that is required for the specification of dorsal midline fates in the Drosophila early embryo. TSG is unlike other secreted growth and differentiation factors in Drosophila in that its expression pattern can be experimentally varied and still result in normal development. Because of this, its regulatory region may be freer to diverge than that of other developmental genes whose misexpression may lead to lethal defects. Thus, the tsg gene may be a good indicator of the frequency and nature of evolutionary changes affecting patterns of gene expression. Over ∼60 million years (Myr), the tsg gene has retained a dorsal-on/ventral-off pattern and a middorsal region of expression; but there have been marked changes in the middorsal domain of expression as well as the appearance/loss of other domains of expression along the anterior/posterior axis. Changes between closely related species (∼2–5 Myr since divergence) that are not reflected among more distantly related species suggest frequent changes in gene expression over evolutionary time. These changes in gene expression may serve as the raw material for eventual evolutionary changes in morphology. Received: 24 March 1997 / Accepted: 20 June 1997     

Convergent, modular expression of ebony and tan in the mimetic wing patterns of Heliconius butterflies

The evolution of pigmentation in vertebrates and flies has involved repeated divergence at a small number of genes related to melanin synthesis. Here, we study insect melanin synthesis genes in Heliconius butterflies, a group characterised by its diversity of wing patterns consisting of black (melanin), and yellow and red (ommochrome) pigmented scales. Consistent with their respective biochemical roles in Drosophila melanogaster, ebony is upregulated in non-melanic wing regions destined to be pigmented red whilst tan is upregulated in melanic regions. Wing regions destined to be pigmented yellow, however, are downregulated for both genes. This pattern is conserved across multiple divergent and convergent phenotypes within the Heliconii, suggesting a conserved mechanism for the development of black, red and yellow pattern elements across the genus. Linkage mapping of five melanin biosynthesis genes showed that, in contrast to other organisms, these genes do not control pattern polymorphism. Thus, the pigmentation genes themselves are not the locus of evolutionary change but lie downstream of a wing pattern regulatory factor. The results suggest a modular system in which particular combinations of genes are switched on whenever red, yellow or black pattern elements are favoured by natural selection for diverse and mimetic wing patterns.     

Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns

BACKGROUND: Animal melanin patterns are involved in diverse aspects of their ecology, from thermoregulation to mimicry. Many theoretical models have simulated pigment patterning, but little is known about the developmental mechanisms of color pattern formation. In Drosophila melanogaster, several genes are known to be necessary for cuticular melanization, but the involvement of these genes in melanin pattern evolution is unknown. We have taken a genetic approach to elucidate the developmental mechanisms underlying melanin pattern formation in various drosophilids. RESULTS: We show that, in D. melanogaster, tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are required for melanin synthesis. Ectopic expression of TH, but not DDC, alone was sufficient to cause ectopic melanin patterns in the wing. Thus, changes in the level of expression of a single gene can result in a new level of melanization. The ontogeny of this ectopic melanization resembled that found in Drosophila species bearing wing melanin patterns and in D. melanogaster ebony mutants. Importantly, we discovered that in D. melanogaster and three other Drosophila species these wing melanin patterns are dependent upon and shaped by the circulation patterns of hemolymph in the wing veins. CONCLUSIONS: Complex wing melanin patterns are determined by two distinct developmental mechanisms. Spatial prepatterns of enzymatic activity are established late in wing development. Then, in newly eclosed adults, melanin precursors gradually diffuse out from wing veins and are oxidized into dark brown or black melanin. Both the prepatterning and hemolymph-supplied components of this system can change during evolution to produce color pattern diversity.     

Expression Pattern Diversity and Functional Conservation Between Retroposed PRAT Genes from Drosophila melanogaster and Drosophila virilis

Gene duplication by retrotransposition duplicates only the coding and untranslated regions of a gene and, thus, biases retroduplicated genes toward having different expression patterns from their parental genes. As such, genes duplicated by retrotransposition are more likely to develop novel expression domains. To explore this idea further, we used the Prat/Prat2 gene duplication in Drosophila as a case study to examine the aftermath of a retrotransposition event that resulted in both the parent and the child gene becoming essential for survival. We used the Gal4-UAS transgene system with EGFP as a reporter to determine the developmental expression patterns of Prat and Prat2 from D. melanogaster (DmPrat and DmPrat2) and Prat from D. virilis (DvPrat). We also tested the functional equivalence of the protein products of DmPrat and DmPrat2. We found that each of the proteins could rescue DmPrat mutations, showing that the requirement for both Prat and Prat2 in Drosophila is not simply due to differences in protein function. In contrast, we found that the DmPrat and DmPrat2 genes have developed nonoverlapping patterns of expression, which correlate with their respective loss-of-function phenotypes. We further found that DvPrat expression is similar to DmPrat during development but differs in adult gonads. Thus, the function of the Prat retrogene has not diverged in the D. melanogaster and D. virilis lineages, while some aspects of its expression pattern have evolved. Finally, we have identified promoter elements, conserved upstream of DmPrat and DvPrat, that this retrogene has acquired to drive its expression.     

Evolution of the glucose dehydrogenase gene in Drosophila

The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.     

Evolution acts on enhancer organization to fine-tune gradient threshold readouts

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.     

Compensatory cis-trans evolution and the dysregulation of gene expression in interspecific hybrids of Drosophila

Hybrids between species are often characterized by novel gene-expression patterns. A recent study on allele-specific gene expression in hybrids between species of Drosophila revealed cases in which cis- and trans-regulatory elements within species had coevolved in such a way that changes in cis-regulatory elements are compensated by changes in trans-regulatory elements. We hypothesized that such coevolution should often lead to gene misexpression in the hybrid. To test this hypothesis, we estimated allele-specific expression and overall expression levels for 31 genes in D. melanogaster, D. simulans, and their F1 hybrid. We found that 13 genes with cis-trans compensatory evolution are in fact misexpressed in the hybrid. These represent candidate genes whose dysregulation might be the consequence of coevolution of cis- and trans-regulatory elements within species. Using a mathematical model for the regulation of gene expression, we explored the conditions under which cis-trans compensatory evolution can lead to misexpression in interspecific hybrids.     

Evolution after Whole-Genome Duplication: Teleost MicroRNAs

MicroRNAs (miRNAs) are important gene expression regulators implicated in many biological processes, but we lack a global understanding of how miRNA genes evolve and contribute to developmental canalization and phenotypic diversification. Whole-genome duplication events likely provide a substrate for species divergence and phenotypic change by increasing gene numbers and relaxing evolutionary pressures. To understand the consequences of genome duplication on miRNA evolution, we studied miRNA genes following the teleost genome duplication (TGD). Analysis of miRNA genes in four teleosts and in spotted gar, whose lineage diverged before the TGD, revealed that miRNA genes were retained in ohnologous pairs more frequently than protein-coding genes, and that gene losses occurred rapidly after the TGD. Genomic context influenced retention rates, with clustered miRNA genes retained more often than nonclustered miRNA genes and intergenic miRNA genes retained more frequently than intragenic miRNA genes, which often shared the evolutionary fate of their protein-coding host. Expression analyses revealed both conserved and divergent expression patterns across species in line with miRNA functions in phenotypic canalization and diversification, respectively. Finally, major strands of miRNA genes experienced stronger purifying selection, especially in their seeds and 3′-complementary regions, compared with minor strands, which nonetheless also displayed evolutionary features compatible with constrained function. This study provides the first genome-wide, multispecies analysis of the mechanisms influencing metazoan miRNA evolution after whole-genome duplication.     

The evolution of gene regulation underlies a morphological difference between two Drosophila sister species

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.